IL 10 Human, His
Description
Production and Purification
Recombinant IL-10 Human, His is synthesized using eukaryotic expression systems to ensure proper folding and biological activity:
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Expression hosts: Sf9 Baculovirus cells or mammalian cell lines .
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Purification: Affinity chromatography leveraging the His tag, followed by proprietary polishing steps .
Table 1: Production Systems for IL-10 Human, His
| Parameter | Sf9 Baculovirus System | Mammalian System |
|---|---|---|
| Tag position | C-terminal His tag | N-terminal His tag |
| Glycosylation | Yes | Yes |
| Purity | >95% | >95% |
| Yield | High | Moderate |
Functional Domains and Mechanisms
IL-10 Human, His retains the functional domains of native IL-10:
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Receptor binding: Binds to the IL-10 receptor complex (IL-10R1/IL-10R2), activating JAK1/TYK2-STAT3 signaling .
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Immunosuppressive activity:
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Immunostimulatory potential: Enhances CD8+ T cell cytotoxicity (Granzyme B, Perforin) and IFNγ production in tumor microenvironments .
Anti-inflammatory Studies
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In vitro models: Suppresses LPS-induced IL-8 production in monocytes and inhibits Th1 cytokine secretion .
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Clinical relevance: Tested in trials for Crohn’s disease and psoriasis, though systemic administration showed mixed efficacy due to pleiotropic effects .
Cancer Immunotherapy
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Preclinical data: PEGylated IL-10 (PEG-rHuIL-10) promotes CD8+ T cell-mediated tumor regression in murine models .
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Mechanistic insight: Enhances STAT3-dependent hyaluronan synthesis in fibroblasts, aiding tissue repair .
Table 2: Key Functional Properties of IL-10 Human, His
Challenges and Future Directions
Product Specs
Q&A
How does the structural configuration of His-tagged recombinant human IL-10 influence its functional activity?
Recombinant human IL-10 is produced as a homodimer stabilized by two intrachain disulfide bonds, with each monomer comprising six α-helices . The addition of a His tag facilitates purification via immobilized metal affinity chromatography (IMAC) but may alter conformational dynamics. Researchers must validate bioactivity post-purification using assays such as:
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Proliferation assays with IL-10-responsive B-cell lines (e.g., MC/9 murine mast cells)
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Cytokine suppression tests measuring IL-10-mediated inhibition of TNF-α or IL-6 in macrophages
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Surface plasmon resonance (SPR) to compare binding kinetics of His-tagged and native IL-10 to the IL-10 receptor (IL-10R)
Tag placement (N- vs. C-terminal) can affect receptor binding. For example, C-terminal tags may sterically hinder interactions with IL-10Rα subunits, reducing signaling efficacy . Always include tagless IL-10 as a control in functional studies.
What methodologies ensure accurate quantification of low-abundance IL-10 in human serum?
The Human IL-10 Quantikine HS ELISA Kit (R&D Systems HS100C) demonstrates a sensitivity of 0.22 pg/mL, making it suitable for detecting physiological IL-10 levels (typically <10 pg/mL in healthy serum) . Key validation parameters include:
| Parameter | Intra-Assay (n=20) | Inter-Assay (n=35) |
|---|---|---|
| Mean (pg/mL) | 2.36–21 | 1.68–20.9 |
| CV% | 4.6–9.4 | 7.8–12.8 |
| Recovery (%) | 81–108 | - |
Methodological recommendations:
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Use citrate plasma over EDTA plasma for 7% higher recovery rates
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Pre-clear samples with protein G columns to remove heterophilic antibodies
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Spike-and-recovery experiments to validate matrix effects (e.g., 93% recovery in citrate plasma)
How do IL-10’s dual immunostimulatory and immunosuppressive roles impact experimental design?
IL-10 exhibits context-dependent duality:
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Anti-inflammatory: Suppresses MHC-II and CD80/86 on dendritic cells, reducing Th1/Th17 activation
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Pro-inflammatory: Enhances CD8+ T-cell cytotoxicity and B-cell survival
Experimental strategies:
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Temporal controls: Measure IL-10 at multiple timepoints (e.g., days 0/3/6 post-stimulation)
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Cell-type-specific knockdowns: Use Cre-lox systems to delete IL-10Rα in myeloid vs. lymphoid lineages
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Combination biomarkers: Pair IL-10 with IL-6 or IL-1RA to predict clinical outcomes (AUC = 0.89 in COVID-19)
What mechanisms underlie contradictory IL-10 expression data across autoimmune models?
Discrepancies arise from:
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Genetic modifiers: IL-10 promoter polymorphisms (e.g., -592C/A) causing 5-fold expression variability
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Disease phase: Elevated IL-10 in early lupus (protective) vs. late-stage disease (pathogenic)
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Compartmentalization: Serum vs. tissue IL-10 levels show poor correlation (r = 0.32 in rheumatoid arthritis)
Resolution workflow:
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Stratify patients by IL-10Rβ expression quartiles
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Perform single-cell RNA-seq to identify IL-10-producing subsets (e.g., Tr1 vs. Bregs)
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Validate using IL-10 reporter mice (e.g., IL-10eGFP)
How can researchers optimize IL-10 detection in post-mortem tissue samples?
Challenges: Protein degradation (t<sub>1/2</sub> = 2.1 hr in vivo) , epitope masking in formalin-fixed tissues.
Solutions:
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Stabilization: Add protease inhibitors (e.g., 1 mM PMSF) within 30 min of collection
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Antigen retrieval: Use pH 9.0 Tris-EDTA buffer with 0.05% Tween-20
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Multiplex IHC: Co-stain for IL-10 and lineage markers (CD68 for macrophages, CD19 for B cells)
What statistical approaches address high inter-individual variability in IL-10 studies?
Always report coefficient of variation (CV%) for assay replicates and adjust for confounders like age (IL-10 decreases 3.2% per decade) and BMI (r = 0.41 with adipocyte-derived IL-10) .
How does IL-10’s role in the JAK-STAT pathway inform inhibitor selection?
IL-10 signaling activates JAK1/TYK2, phosphorylating STAT3 homodimers that upregulate SOCS3 (a negative feedback regulator) . When testing JAK inhibitors:
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Tofacitinib: 50 nM IC50 for JAK1, reduces IL-10-mediated pSTAT3 by 78%
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Ruxolitinib: Sparing effect on TYK2 preserves 40% IL-10 anti-inflammatory activity
Dosing considerations:
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Pre-treat cells with inhibitors 1 hr pre-IL-10 stimulation
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Monitor SOCS3 knockdown (siRNA) to amplify IL-10 effects
What translational insights emerge from IL-10’s ACE2 regulatory role in viral infections?
IL-10 upregulates ACE2 via STAT3, potentially mitigating COVID-19 severity by:
Therapeutic implications:
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IL-10 infusion (10 μg/kg) in SARS-CoV-2-infected mice decreases lung viral load by 2.1 log<sub>10</sub>
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Correlate serum IL-10 with soluble ACE2 (r = 0.67) to identify responsive patients
How do IL-10’s neuroimmunological functions inform CNS disease models?
Key findings:
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Neuroprotection: IL-10 reduces infarct volume by 32% in stroke models
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Axon regeneration: Schwann cell-derived IL-10 increases neurite outgrowth by 41%
Model optimization:
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Use GFAP-IL-10 transgenic mice to target astrocyte-specific overexpression
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Combine with RNAscope™ to localize IL-10 mRNA in microglial subsets
What criteria define clinically relevant IL-10 thresholds in cancer immunotherapy?
Thresholds vary by malignancy:
| Cancer Type | Prognostic Cutoff | Hazard Ratio |
|---|---|---|
| Melanoma | >15 pg/mL serum | 2.1 (95% CI: 1.6–2.8) |
| Colorectal | >8 pg/mL tumor | 0.6 (95% CI: 0.4–0.9) |
Mechanistic basis:
Product Science Overview
Interleukin-10, also known as human cytokine synthesis inhibitory factor, is an anti-inflammatory cytokine. It is primarily produced by monocytes and, to a lesser extent, by lymphocytes. Interleukin-10 plays a crucial role in immunoregulation and inflammation by down-regulating the expression of Th1 cytokines, major histocompatibility complex class II antigens, and costimulatory molecules on macrophages .
Human recombinant interleukin-10 is typically produced using an expression system in Escherichia coli. The protein is often tagged with a histidine tag at the C-terminus to facilitate purification. The recombinant protein is then lyophilized from a filtered solution of phosphate-buffered saline at pH 8.0. The final product is highly pure, with a purity greater than 98% as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis .
Interleukin-10 signals through a receptor complex consisting of two interleukin-10 receptor-1 and two interleukin-10 receptor-2 proteins. The binding of interleukin-10 to its receptor induces signal transducer and activator of transcription 3 signaling via the phosphorylation of the cytoplasmic tails of interleukin-10 receptor 1 and interleukin-10 receptor 2 by Janus kinase 1 and tyrosine kinase 2, respectively . This signaling pathway is essential for the anti-inflammatory effects of interleukin-10, as it inhibits the production of pro-inflammatory cytokines and limits the immune response .
Interleukin-10 is a key immunoregulator during infection due to its inhibitory effect on inflammatory cytokine production. It helps suppress excessive type 1 helper T cell and CD8+ T cell responses during infection, thereby preventing tissue damage and maintaining immune homeostasis .
